Polynucleotides for the amplification and detection of chlamydia trachomatis and neisseria gonorrhoeae

ABSTRACT

Polynucleotides useful for detecting  Chlamydia trachomatis  and/or  Neisseria gonorrhoeae  in a test sample, kits, a nucleic acid amplification method and detection method including the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 12/764,301,filed on Apr. 21, 2010, which is a divisional of U.S. patent applicationSer. No. 10/292,420, filed on Nov. 12, 2002, now issued as U.S. Pat. No.7,807,802, the contents of all of which are fully incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 6, 2014, isnamed 6873USD2_SL.txt and is 3,255 bytes in size.

FIELD OF THE INVENTION

The present invention relates to Chlamydia trachomatis and Neisseriagonorrhoeae. In particular the invention relates to polynucleotides andmethods for amplifying and detecting Chlamydia trachomatis and Neisseriagonorrhoeae.

BACKGROUND OF THE INVENTION

Chlamydia trachomatis (C. trachomatis or CT) and Neisseria gonorrhoeae(N. gonorrhoeae or NG) are the causative agents of common sexuallytransmitted diseases. CT causes venereal lymphogranuloma, variousinflammatory pathologies of the male and female urogenital systems, andtrachoma, a chronic disease that affects 500 million people and can leadto blindness. When not precociously diagnosed and treated by adequatetherapy, CT-induced urethritis and cervicitis may led to a variety ofchronic inflammations, such as, e.g., vaginitis, salpingitis and pelvicinflammation which may result in sterility and extrauterine pregnancy.Furthermore, the new born from infected mothers may contract pulmonaryand/or ocular infections during delivery.

N. gonorrhoeae, the pathogen of gonorrhoea, manifests itself as apurulent inflammation and swelling of the urethra in males. Thesesymptoms occur in 90% of cases of infection. If left untreated, theinfection can ascend and after several weeks produce symptoms ofprostatitis. In women, no or only slight symptoms occur in 50% of casesof infection. The infection primarily affects the cervix, but also theurethra. In 10 to 15% of women, the infection spreads to the fallopiantubes and can also lead to sterility. Since the course of the infectionsis often asymptomatic, many carriers contribute unknowingly to thespread of the disease.

Considering the impact that these two organisms have, rapid and specificdiagnostic tests are of utmost importance. Diagnosis based on selectivegrowth of the pathogenic bacteria has been the standard, but cellculturing is time-consuming and many clinical isolates are difficult togrow in vitro. Infection with bacteria results in the formation of avariety of antibodies with serogroup, species, subspecies, serovar(serotype) and auxotype specificity. Sera from patients with genitaltract infections have been used to diagnose CT and NG infection, howeverassays based on serological markers are by nature non-quantitative andsubject to difficulties in interpretation. For example, antibody titresmay be undetectable in acute infections (false negative), may persist inuninfected individuals with a past history of infection (falsepositive), may yield a false-positive indication due to the presence ofcross-reacting species (e.g., respiratory infection by differentChlamydia species), or may not develop at all (false negative) dependingon other factors (Ngeow, 1996, Ann Acad Med Singapore 25:300; Black etal., 1991, J Clin Microbiol 29:1312). For these reasons, serology aloneis inadequate for the diagnosis of CT and NG infections.

Bacterial infections may also be diagnosed by the detection of nucleicacid sequences particular to the infectious organism. Depending on thenucleic acid sequence selected for detection, a diagnostic assay may bespecific for an entire genus, more than one genus, a species orsubspecies, auxotype, serovar (serotype), strain or other subset oforganisms. This selectivity may be exploited in the development ofsimple reliable diagnostic tests for C. trachomatis and N. gonorrhoeaespecies of bacterial pathogens.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention. Publications referred to throughout thespecification are herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention provides polynucleotide reagents useful for thedetection of Chlamydia trachomatis and/or Neisseria gonorrhoeae. Inparticular, the present invention provides primers and probes fornucleic acid amplification and detection procedures, which specificallyand sensitively detect various subspecies or serotypes and auxotypes ofC. trachomatis and/or N. gonorrhoeae.

Two or more of the polynucleotide reagents provided herein as SEQ IDNOs: 1-12 can be combined in a composition, such as SEQ ID NO: 1 and oneor more of SEQ ID NOs: 4-7 or such as SEQ ID NO: 2 and one or more ofSEQ ID NOs: 4-7. Similarly, two more of the polynucleotide reagentsprovided herein can be combined such as one or more of SEQ ID NOs: 8 andone of SEQ ID NOs: 9-12, or SEQ ID NO: 9 and one of SEQ ID NOs: 8 and10-12. In certain preferred embodiments, the above compositions comprisea first polynucleotide having a sequence selected from SEQ ID NOs: 1-3,a second polynucleotide having the sequence set forth in SEQ ID NO: 4, athird polynucleotide having the sequence set forth in SEQ ID NO: 8, anda fourth polynucleotide having the sequence set forth in SEQ ID NO: 9.

The primer sets herein provided comprise two primers, and are useful forthe amplification of target sequences, e.g., in PCR. The primersdesignated SEQ ID NOs: 1-3 and SEQ ID NO: 4 specifically amplify C.trachomatis, and the primers designated SEQ ID NO: 8 and SEQ ID NO: 9amplify serotypes and/or auxotypes of N. gonorrhoeae. The sequencesidentified herein as SEQ ID NOs: 5-7 are probe sequences useful fordetecting amplified Chlamydia trachomatis, e.g., when Chlamydiatrachomatis is amplified by primers having sequences identified by SEQID NOs: 1-3 and 4. Similarly, the sequences identified herein as SEQ IDNOs: 10-12 are useful for detecting amplified N. gonorrhoeae, e.g., whenN. gonorrhoeae is amplified by primers having sequences identified bySEQ ID NOs: 8 and 9. Preferably, the primer and probe set comprises twoprimer sequences and one probe sequence. More preferably, the primer andprobe set are used for PCR. Specific primer/probe sets that can beemployed to amplify and detect C. trachomatis include primer/probe sets1-9, which are set forth below. Similarly, specific primer/probe setsthat can be employed to amplify and detect N. gonorrhoeae includeprimer/probe sets 10-12 which are set forth below.

The method for amplifying C. trachomatis and/or N. gonorrhoeae willgenerally comprise (a) forming a reaction mixture comprising nucleicacid amplification reagents, at least one polynucleotide of theinvention, or composition, or primer set, or primer/probe set asmentioned above, and a test sample potentially containing at least onetarget sequence and (b) subjecting the mixture to amplificationconditions to generate at least one copy of a nucleic acid sequencecomplementary to the target sequence.

The method for detecting C. trachomatis and/or N. gonorrhoeae willgenerally comprise (a) forming a reaction mixture comprising nucleicacid amplification reagents, at least one polynucleotide of theinvention, or composition, or primer set, or primer/probe set asmentioned above, and a test sample potentially containing at least onetarget sequence; (b) subjecting the mixture to amplification conditionsto generate at least one copy of a nucleic acid sequence complementaryto the target sequence; (c) hybridizing the probe to the nucleic acidsequence complementary to the target sequence, so as to form a hybridcomprising the probe and the nucleic acid sequence complementary to thetarget sequence; and (d) detecting the hybrid as an indication of thepresence of C. trachomatis and/or N. gonorrhoeae in the test sample.

Further, when the amplification is PCR, or a similar thermocyclingamplification process, step (b) can be repeated multiple times toincrease the number of target sequence copies. According to anotherembodiment, both C. trachomatis and N. gonorrhoeae can be detected in asingle reaction mixture using a combination of the primer/probe sets,especially when a probe having SEQ ID NO: 5, 6, or 7 carries a labelthat produces a signal distinct from the signal generated by a probehaving SEQ ID NO: 10, 11, or 12, and one of each set is contacted to atest sample amplified with SEQ ID NOs: 1-3 and 4, in combination withSEQ ID NOs: 8 and 9.

The polynucleotides of the present invention also can be provided aspart of a kit useful for amplifying and for detecting C. trachomatisand/or N. gonorrhoeae. The kits comprise one or more suitable containerscontaining one or more primer sets or primer/probe sets or primer/probecombinations according to the present invention, an enzyme havingpolymerase activity and deoxynucleotide triphosphates. At least onesequence preferably bears a label.

In another aspect of the invention, a control target polynucleotide anda control polynucleotide probe are included in any of the methods orkits provided herein. In this aspect of the invention, the primer setpreferably is complementary to the target sequence as well as thecontrol target, whereas the target probe is preferably substantiallycomplementary only to the target polynucleotide sequence and the controlprobe is preferably substantially complementary only to the controltarget.

Another aspect of the invention, provides isolated polynucleotideshaving a nucleotide sequence consisting essentially of, and preferablyconsisting of, the nucleotide sequences set forth in SEQ ID NOs: 2-12.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides polynucleotides that can specificallyhybridise with a nucleic acid sequence, or complement thereof, ofChlamydia trachomatis (C. trachomatis or CT) and further provides otherpolynucleotides that can specifically hybridise with a nucleic acidsequence, or complement thereof, of Neisseria gonorrhoeae (N.gonorrhoeae or NG). These polynucleotides can be used to amplifyChlamydia trachomatis and N. gonorrhoeae, and to specifically detect thepresence of each organism to the exclusion of the other. Presently,sixteen strains of Chlamydia trachomatis and 57 auxotypes and serovarsof N. gonorrhoeae are known, and the method suitably and specificallydetects all known strains.

The term “specifically hybridise” as used herein refers to the abilityof a nucleic acid to bind detectably and specifically to a secondnucleic acid. Polynucleotides specifically hybridise with target nucleicacid strands under hybridisation and wash conditions that minimiseappreciable amounts of detectable binding to non-specific nucleic acids.Stringent conditions that can be used to achieve specific hybridisationare known in the art.

A “target sequence” or “target nucleic acid sequence” as used hereinmeans a nucleic acid sequence of CT or NG, or complement thereof, thatis amplified, detected, or both amplified and detected using one or moreof the polynucleotides herein provided. Additionally, while the termtarget sequence sometimes refers to a double stranded nucleic acidsequence, those skilled in the art will recognize that the targetsequence can also be single stranded. In cases where the target isdouble stranded, polynucleotide primer sequences of the presentinvention preferably will amplify both strands of the target sequence. Atarget sequence may be selected that is more or less specific for aparticular organism. For example, the target sequence may be specific toan entire genus, to more than one genus, to a species or subspecies,serogroup, auxotype, serotype, strain, isolate or other subset oforganisms. The polynucleotide sequences of the present invention areselected for their ability to specifically hybridize with a range ofsubspecies, or serotypes or auxotypes, of CT and NG.

The term “test sample” as used herein, means a sample taken from anorganism or biological fluid that is suspected of containing orpotentially contains a CT or NG target sequence. The test sample can betaken from any biological source, such as for example, tissue, blood,saliva, sputa, mucus, sweat, urine, urethral swabs, cervical swabs,urogenital or anal swabs, conjunctival swabs, ocular lens fluid,cerebral spinal fluid, milk, ascites fluid, synovial fluid, peritonealfluid, amniotic fluid, fermentation broths, cell cultures, chemicalreaction mixtures and the like. The test sample can be used (i) directlyas obtained from the source or (ii) following a pre-treatment to modifythe character of the sample. Thus, the test sample can be pre-treatedprior to use by, for example, preparing plasma or serum from blood,disrupting cells or viral particles, preparing liquids from solidmaterials, diluting viscous fluids, filtering liquids, distillingliquids, concentrating liquids, inactivating interfering components,adding reagents, purifying nucleic acids, and the like.

The term “label” as used herein means a molecule or moiety having aproperty or characteristic which is capable of detection and,optionally, of quantitation. A label can be directly detectable, aswith, for example (and without limitation), radioisotopes, fluorophores,chemiluminophores, enzymes, colloidal particles, fluorescentmicroparticles and the like; or a label may be indirectly detectable, aswith, for example, specific binding members. It will be understood thatdirectly detectable labels may require additional components such as,for example, substrates, triggering reagents, quenching moieties, light,and the like to enable detection and/or quantitation of the label. Whenindirectly detectable labels are used, they are typically used incombination with a “conjugate”. A conjugate is typically a specificbinding member that has been attached or coupled to a directlydetectable label. Coupling chemistries for synthesizing a conjugate arewell known in the art and can include, for example, any chemical meansand/or physical means that does not destroy the specific bindingproperty of the specific binding member or the detectable property ofthe label. As used herein, “specific binding member” means a member of abinding pair, i.e., two different molecules where one of the moleculesthrough, for example, chemical or physical means specifically binds tothe other molecule. In addition to antigen and antibody specific bindingpairs, other specific binding pairs include, but are not intended to belimited to, avidin and biotin; haptens and antibodies specific forhaptens; complementary nucleotide sequences; enzyme cofactors orsubstrates and enzymes; and the like.

A polynucleotide, in the context of the present invention, is a nucleicacid polymer of ribonucleic acid (RNA), deoxyribonucleic acid (DNA),modified RNA or DNA, or RNA or DNA mimetics (such as, without limitationPNAs), and derivatives thereof, and homologues thereof. Thus,polynucleotides include polymers composed of naturally occurringnucleobases, sugars and covalent internucleoside (backbone) linkages aswell as polymers having non-naturally-occurring portions that functionsimilarly. Such modified or substituted nucleic acid polymers are wellknown in the art and for the purposes of the present invention, arereferred to as “analogues.” For ease of preparation and familiarity tothe skilled artisan, polynucleotides are preferably modified orunmodified polymers of deoxyribonucleic acid or ribonucleic acid.

Polynucleotide analogues that are useful in the present inventioninclude polymers with modified backbones or non-natural internucleosidelinkages. In accordance with the present invention, modified backbonesinclude those retaining a phosphorus atom in the backbone, such asphosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates, as well as those no longer having a phosphorus atom, suchas backbones formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages. An example of such a non-phosphorus containingbackbone is a morpholino linkage (see, for example, U.S. Pat. Nos.5,185,444, 5,034,506, and 5,142,047 all of which are herein incorporatedby reference). As is known in the art, modified nucleic acid polymers(analogues) may contain one or more modified sugar moieties. Forexample, sugar moieties may be modified by substitution at the 2′position with a 2-methoxyethoxy (2-MOE) group (see, for example, Martinet al., (1995) Helv. Chim. Acta, 78:486-504).

The present invention also contemplates analogues that are RNA or DNAmimetics, in which both the sugar and the internucleoside linkage of thenucleotide units are replaced with novel groups. In these mimetics thebase units are maintained for hybridisation with the target sequence. Anexample of such a mimetic, which has been shown to have excellenthybridisation properties, is a peptide nucleic acid (PNA) (Nielsen etal., (1991) Science, 254:1497-1500; International Patent Application WO92/20702, herein incorporated by reference). In PNA compounds, thesugar-backbone of an oligonucleotide is replaced with an amidecontaining backbone, for example an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to theaza-nitrogen atoms of the amide portion of the backbone.

Contemplated polynucleotides of the invention further includederivatives wherein the nucleic acid molecule has been covalentlymodified by substitution, chemical, enzymatic, or other appropriatemeans with a moiety other than a naturally occurring nucleotide, forexample with a moiety that functions as a label, as described herein.

The present invention further encompasses homologues of thepolynucleotides having nucleic acid sequences set forth in SEQ ID NOs:1-12. Homologues are nucleic acids having at least one alteration in theprimary sequence set forth in any one of SEQ ID NOs: 1-12, that does notdestroy the ability of the polynucleotide to specifically hybridise witha target sequence, as described above. Accordingly, a primary sequencecan be altered, for example, by the insertion, addition, deletion orsubstitution of one or more of the nucleotides of, for example, SEQ IDNOs: 1-12. Thus, homologues that are fragments of a sequence disclosedin SEQ ID NOs: 1-12 may have a consecutive sequence of at least about 7,10, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23 or more nucleotides of thenucleic acid sequences of SEQ ID NO: 1-12, and will retain the abilityto specifically hybridise with a target sequence, as described above.Ordinarily, the homologues will have a nucleic acid sequence having atleast about 50%, 60%, 70%, 80%, 85%, 90% or 95% nucleic acid sequenceidentity with a nucleic acid sequence set forth in SEQ ID NOs: 1-12.Identity with respect to such sequences is defined herein as thepercentage of nucleotides in the candidate sequence that are identicalwith the known polynucleotides after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent identity.Terminal (5′ or 3′) or internal deletions, extensions or insertions intothe nucleotide sequence shall not be construed as affecting identity.

The polynucleotides of the present invention thus comprise primers andprobes that specifically hybridize to a target sequence of theinvention, for example the nucleic acid molecules having any one of thenucleic acid sequences set forth in SEQ ID NOs: 1-12, includinganalogues and/or derivatives of said nucleic acid sequences, andhomologues thereof, that can specifically hybridise with a targetsequence of the invention. As described below, polynucleotides of theinvention can be used as primers and/or probes to amplify or detectChlamydia trachomatis or N. gonorrhoeae.

Such hybridizing polynucleotides, however, are defined further as beingnovel and non-obvious over any prior art polynucleotide including thatwhich hybridizes under appropriate stringency conditions, a targetsequence according to the present invention.

The polynucleotides according to the present invention can be preparedby conventional techniques well known to those skilled in the art. Forexample, the polynucleotides can be prepared using conventionalsolid-phase synthesis using commercially available equipment, such asthat available from Applied Biosystems USA Inc. (Foster City, Calif.),DuPont, (Wilmington, Del.), or Milligen (Bedford, Mass.). Modifiedpolynucleotides, such as phosphorothioates and alkylated derivatives,can also be readily prepared by similar methods known in the art. See,for example, U.S. Pat. Nos. 5,464,746; 5,424,414; and 4,948,882.

The polynucleotides according to the present invention can be employeddirectly as probes for the detection, or quantitation, or both, of CTand/or NG nucleic acids in a test sample. The test sample is contactedwith at least one of the polynucleotides of the present invention undersuitable hybridisation conditions and the hybridization between thetarget sequence and at least one of the polynucleotides is then detectedby methods well-known in the art.

The polynucleotides of the present invention may incorporate one or moredetectable labels. Detectable labels are molecules or moieties having aproperty or characteristic that can be detected directly or indirectlyand are chosen such that the ability of the polynucleotide to hybridisewith its target sequence is not adversely affected. Methods of labelingnucleic acid sequences are well known in the art (see, for example,Ausubel et al., (1997 & updates) Current Protocols in Molecular Biology,Wiley & Sons, New York).

Detection labels have the same definition as “labels” previously definedand “capture labels” are typically used to separate extension products,and probes associated with any such products, from other amplificationreactants. Specific binding members (as previously defined) are wellsuited for this purpose. Also, probes used according to this method maybe blocked at their 3′ ends so that they are not extended underhybridization conditions. Methods for preventing extension of a probeare well known and are a matter of choice for one skilled in the artTypically, adding a phosphate group to the 3′ end of the probe willsuffice for purposes of blocking extension of the probe.

In cases where labels are employed to detect primer-amplified products,primer sequences optionally can be labeled with either a capture labelor a detection label. The probe sequence is used to hybridize with thesequence generated by the primer sequence, and typically hybridizes witha sequence that does not include the primer sequence. Similarly to theprimer sequence, the probe sequence can also labeled with either acapture label or a detection label with the caveat that when the primeris labeled with a capture label, the probe is labeled with a detectionlabel, and vice versa. Upon formation of the copy sequence/probehybrids, the differential labels (i.e., capture and detection labels) onthe copy sequence and probe sequence can be used to separate and detectsuch hybrids. In one embodiment of the present invention, detection isperformed according to the protocols used by the commercially availableAbbott LCx® instrumentation (Abbott Laboratories; Abbott Park, Ill.).

The polynucleotides according to the present invention are also suitablefor use as capture probes in sandwich-type assays. Capture probes andsandwich hybridisation assays are well known in the art. Briefly, thepolynucleotide capture probe is attached to a solid support and broughtinto contact with a test sample under suitable hybridisation conditionssuch that a probe:target hybrid is formed between the capture probe andany target nucleic acid present in the test sample. After one or moreappropriate washing steps, the probe:target hybrid is detected, usuallyby means of a second “disclosure” probe or by a specific antibody thatrecognises the hybrid molecule. The use of the CT and/or NGpolynucleotides of the invention as a capture or a disclosure probe, orboth, in such sandwich hybridisation assays is thus considered to bewithin the scope of the present invention.

The present invention also contemplates the use of the polynucleotidesin modified nucleic acid hybridisation assays. For example, U.S. Pat.No. 5,627,030 discloses a method to amplify the detection signal in anucleic acid hybridisation assay. In the disclosed assay, a firstpolynucleotide probe sequence is hybridised under suitable conditions toa target sequence, the probe:target hybrid is subsequentlyimmunocaptured and immobilised. A second polynucleotide probe whichcontains many repeating sequence units is then hybridised to the probecomponent of the probe:target hybrid. Detection is achieved byhybridisation of many labeled nucleic acid sequence probes, one to eachof the repeating sequence units present in the second probe. Theattachment of multiple labeled probes to the second probe thus amplifiesthe detection signal and increases the sensitivity of the assay. The useof the polynucleotides of the instant invention in modifiedhybridisation assays of this type, either directly as a first probe, oras a second probe after modification to incorporate additional repeatingsequence units by standard techniques, is thus considered to be withinthe scope of the present invention.

i) Amplification and Detection of CT and/or NG Nucleotide Sequences

The polynucleotides of the invention can be used as primers or probes toamplify and/or detect CT or NG in a test sample. The primer/probe setsprovided herein comprise two primers and at least one probe. Theseprimer/probe sets can be employed according to nucleic acidamplification techniques. Hence, the primers in any particularprimer/probe set can be employed to amplify a target sequence. In mostcases, the probe hybridizes to the copies of the target sequencegenerated by one of the primers and generally facilitates detecting anycopies of the target sequence generated during the course of theamplification reaction. All of the primer/probe sets can be employedaccording to nucleic acid amplification procedures to specifically andsensitively detect either CT or NG, or both CT and NG when theappropriate primers and probes are combined. It is contemplated that theindividual primers and probes of the primer/probe sets provided hereinmay alternatively be used in combination with primers and/or probesother than those described in the primer/probe sets provided herein.

Amplification procedures are well-known in the art and include, but arenot limited to, polymerase chain reaction (PCR), TMA, rolling circleamplification, nucleic acid sequence based amplification (NASBA), andstrand displacement amplification (SDA). One skilled in the art willunderstand that for use in certain amplification techniques the primersmay need to be modified, for example, for SDA the primer comprisesadditional nucleotides near its 5′ end that constitute a recognitionsite for a restriction endonuclease. Similarly, for NASBA the primercomprises additional nucleotides near the 5′ end that constitute an RNApolymerase promoter. Polynucleotides thus modified are considered to bewithin the scope of the present invention.

As is well known in the art, certain criteria need to be taken intoconsideration when selecting a primer for an amplification reaction. Forexample, when a primer pair is required for the amplification reaction,the primers should be selected such that the likelihood of forming 3′duplexes is minimised, and such that the melting temperatures (T_(M))are sufficiently similar to optimise annealing to the target sequenceand minimise the amount of non-specific annealing. In this context, thepolynucleotides according to the present invention are provided incombinations that can be used as primers in amplification reactions tospecifically amplify target nucleic acid sequences.

In one embodiment of the present invention, therefore, polynucleotideshaving the nucleic acid sequences as set forth in SEQ ID NOs: 1, 2, or 3used in combination with the nucleic acid sequences as set forth in SEQID NO: 4, which are provided with polynucleotides having the nucleicacid sequences as set forth in SEQ ID NOs: 8 and 9. In a relatedembodiment, these primer combinations are used to specifically amplifyCT and/or NG nucleic acid sequences, if present in a test sample.Primers included in the primer/probe sets 1-9 (further defined below)can be used to prime synthesis and amplification of copies of a CTtarget sequence in the case of SEQ ID NOs: 1, 2, or 3 and 4; and copiesof a NG target sequence in the case of primer/probe sets 10-12 and SEQID NOs: 8 and 9. The remaining SEQ ID NOs (SEQ ID NOs: 5-7 and 10-12)can specifically hybridize with the amplification products of the primersequences found in the same primer/probe set. For example, primer/probeset 10 is specific for NG insofar as SEQ ID NOs: 8 and 9 prime synthesisof the NG target sequence and SEQ ID NO: 10 hybridizes with theamplification products produced by SEQ ID NOs: 8 and 9.

The amplification method of the present invention generally comprises(a) forming a reaction mixture comprising nucleic acid amplificationreagents, at least one primer/probe set of the present invention, and atest sample suspected of containing a at least one target sequence and(b) subjecting the mixture to amplification conditions to generate atleast one copy of a nucleic acid sequence complementary to the targetsequence.

Step (b) of the above methods can be repeated any suitable number oftimes (prior to step (c) in the detection method), e.g., by thermalcycling the reaction mixture between 10 and 100 times, typically betweenabout 20 and about 60 times, more typically between about 25 and about45 times.

Nucleic acid amplification reagents include reagents which are wellknown and may include, but are not limited to, an enzyme having at leastpolymerase activity, enzyme cofactors such as magnesium or manganese;salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotidetriphosphates (dNTPs) such as for example deoxyadenine triphosphate,deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythyminetriphosphate.

Amplification conditions are conditions that generally promote annealingand extension of one or more nucleic acid sequences. It is well knownthat such annealing is dependent in a rather predictable manner onseveral parameters, including temperature, ionic strength, sequencelength, complementarity, and G:C content of the sequences. For example,lowering the temperature in the environment of complementary nucleicacid sequences promotes annealing. For any given set of sequences, melttemperature, or Tm, can be estimated by any of several known methods.Typically, diagnostic applications utilize hybridization temperaturesthat are about 10° C. (e.g., 2° C. to 18° C.) below the melttemperature. Ionic strength or “salt” concentration also impacts themelt temperature, since small cations tend to stabilize the formation ofduplexes by negating the negative charge on the phosphodiester backbone.Typical salt concentrations depend on the nature and valency of thecation but are readily understood by those skilled in the art.Similarly, high G:C content and increased sequence length are also knownto stabilize duplex formation because G:C pairings involve 3 hydrogenbonds where A:T pairs have just two, and because longer sequences havemore hydrogen bonds holding the sequences together. Thus, a high G:Ccontent and longer sequence lengths impact the hybridization conditionsby elevating the melt temperature.

Once sequences are selected for a given diagnostic application, the G:Ccontent and length will be known and can be accounted for in determiningprecisely what hybridization conditions will encompass. Since ionicstrength is typically optimized for enzymatic activity, the onlyparameter left to vary is the temperature. Generally, the hybridizationtemperature is selected close to or at the Tm of the primers or probe.Thus, obtaining suitable hybridization conditions for a particularprimer/probe set is well within ordinary skill of one practicing thisart.

As mentioned earlier, the primer sequences (SEQ ID NOs: 1-4, 8 and 9) ofany particular primer/probe set can, by themselves or with additionalpolynucleotides, be used as amplification primers according to nucleicacid amplification procedures well known in the art. Such reactionsinclude, but are not intended to be limited to, the polymerase chainreaction (PCR) described in U.S. Pat. Nos. 4,683,195 and 4,683,202, theligase chain reaction (LCR) described in EP-A-320 308, and gap LCR(GLCR) described in U.S. Pat. No. 5,427,930. Each of these exemplaryamplification reactions generate multiple copies of a DNA targetsequence.

In one embodiment of the present invention, the detection methodgenerally comprises (a) forming a reaction mixture comprising nucleicacid amplification reagents, at least one primer/probe set of thepresent invention, and a test sample suspected of containing at leastone target sequence; (b) subjecting the mixture to amplificationconditions to generate at least one copy of a nucleic acid sequencecomplementary to the target sequence; (c) hybridizing the probe to thenucleic acid sequence complementary to the target sequence, so as toform a hybrid comprising the probe and the nucleic acid sequencecomplementary to the target sequence; and (d) detecting the hybrid as anindication of the presence of the target sequence (CT and/or NG and/or acontrol sequence) in the sample.

Specific amplicons produced by amplification of target nucleic acidsequences using the polynucleotides of the present invention, asdescribed above, can be detected by a variety of methods known in theart. For example, one or more of the primers used in the amplificationreactions may be labeled such that an amplicon can be directly detectedby conventional techniques subsequent to the amplification reaction.Alternatively, a probe consisting of a labeled version of one of theprimers used in the amplification reaction, or a third polynucleotidedistinct from the primer sequences that has been labeled and iscomplementary to a region of the amplified sequence, can be added afterthe amplification reaction is complete. The mixture is then submitted toappropriate hybridisation and wash conditions and the label is detectedby conventional methods.

The amplification product produced as above can be detected during orsubsequently to the amplification of the target sequence. Methods fordetecting the amplification of a target sequence during amplificationare outlined above, and described, for example, in U.S. Pat. No.5,210,015. Gel electrophoresis can be employed to detect the products ofan amplification reaction after its completion. Alternatively,amplification products are hybridized to probes, then separated fromother reaction components and detected using microparticles and labeledprobes.

It will be readily appreciated that a procedure that allows bothamplification and detection of target nucleic acid sequences to takeplace concurrently in a single unopened reaction vessel would beadvantageous. Such a procedure would avoid the risk of “carry-over”contamination in the post-amplification processing steps, and would alsofacilitate high-throughput screening or assays and the adaptation of theprocedure to automation. Furthermore, this type of procedure allows“real-time” monitoring of the amplification reaction as well as moreconventional “end-point” monitoring.

The present invention thus includes the use of the polynucleotides in amethod to specifically amplify and detect target nucleic acid sequencesin a test sample in a single tube format. This may be achieved, forexample, by including in the reaction vessel an intercalating dye suchas SYBR Green or an antibody that specifically detects the amplifiednucleic acid sequence. Alternatively a third polynucleotide distinctfrom the primer sequences, which is complementary to a region of theamplified sequence, may be included in the reaction, as when aprimer/probe set of the invention is used.

For use in an assay as described above, in which both amplification withpolynucleotide primers and detection of target sequences using apolynucleotide probe occur concurrently in a single unopened reactionvessel, the polynucleotide probe needs to possess certain properties.For example, since the probe will be present during the amplificationreaction, it should not interfere with the progress of this reaction andshould also be stable under the reaction conditions. In addition, forreal-time monitoring of reactions, the probe should be capable ofbinding its target sequence under the conditions of the amplificationreaction and to emit a signal only upon binding this target sequence.Examples of probe molecules that are particularly well-suited to thistype of procedure include molecular beacon probes and TaqMan® probes.

The present invention, therefore, contemplates the use of thepolynucleotides as TaqMan® probes. As is known in the art, TaqMan®probes are dual-labeled fluorogenic nucleic acid probes composed of apolynucleotide complementary to the target sequence that is labeled atthe 5′ terminus with a fluorophore and at the 3′ terminus with aquencher. TaqMan® probes are typically used as real-time probes inamplification reactions. In the free probe, the close proximity of thefluorophore and the quencher ensures that the fluorophore is internallyquenched. During the extension phase of the amplification reaction, theprobe is cleaved by the 5′ nuclease activity of the polymerase and thefluorophore is released. The released fluorophore can then fluoresce andthus produces a detectable signal.

The present invention further contemplates the use of thepolynucleotides as “molecular beacon” probes. Molecular beacon probesare well known in the art, for example, see U.S. Pat. Nos: 6,150,097;5,925,517 and 6,103,476. Basically, molecular beacons are polynucleotideprobes capable of forming a stem-loop (hairpin) structure. The loop is asingle-stranded structure containing sequences complementary to thetarget sequence, whereas the stem typically is unrelated to the targetsequence and self-hybridises to form a double-stranded region.Nucleotides that are both complementary to the target sequence and thatcan self-hybridise may also form part of the stem region. Attached toone arm of the stem is a fluorophore moiety and to the other arm aquencher moiety. When the polynucleotide adopts a hairpin shape, thefluorophore and the quencher are in close proximity and the energyemitted by the fluorophore is thus absorbed by the quencher and givenoff as heat, resulting in internal quenching of the fluorophore. Uponbinding of the polynucleotide to its target sequence, the fluorophoreand the quencher become spatially separated and the fluorophore canfluoresce producing a detectable signal. Preferably, the primers andprobes of the present invention are selected such that the probe stably(e.g., less than 5% of probe bound to amplicon is displaced over 24hours under the hybridization conditions for the probe to the amplicon)binds to the amplicon when the reaction is cooled from a denaturationtemperature, e.g., 90° C. to 96° C., to a temperature below the Tm forthe binding of the probe with the amplicon.

The present invention further contemplates the use of polynucleotides ofthe invention as linear probes in conjunction with a fluorophore and ahigh efficiency quencher, such as the Black Hole Quenchers (BHQ™;Biosearch Technologies, Inc., Novato, Calif.). As is known in the art,the high quenching efficiency and lack of native fluorescence of theBHQ™ dyes allows “random-coil” quenching to occur in linear probeslabeled at one terminus with a fluorophore and at the other with a BHQ™dye thus ensuring that the fluorophore does not fluoresce when the probeis in solution. Upon binding its target sequence, the probe stretchesout, the fluorophore and quencher are thus spatially separated and thefluorophore fluoresces. One skilled in the art will appreciate that theBHQ™ dyes can also be used as the quencher moiety in molecular beacon orTaqMan™ probes.

Suitable fluorophores and quenchers for use with the polynucleotides ofthe present invention can be readily determined by one skilled in theart (see also, Tyagi et al., Nature Biotechnol., 16:49-53 (1998); Marraset al., Genet. Anal.: Biomolec. Eng., 14:151-156 (1999)). Manyfluorophores and quenchers are available commercially, for example fromMolecular Probes (Eugene, Oreg.) or Biosearch Technologies, Inc.(Novato, Calif.). Examples of fluorophores that can be used in thepresent invention include, but are not limited to, fluorescein andfluorescein derivatives such as a dihalo-(C₁ toC₈)dialkoxycarboxyfluorescein,5-(2′-aminoethyl)aminonaphthalene-1-sulphonic acid (EDANS), coumarin andcoumarin derivatives, Lucifer yellow, Texas red, tetramethylrhodamine,tetrachloro-6-carboxyfluoroscein, 5-carboxyrhodamine, cyanine dyes andthe like. Quenchers include, but are not limited to, DABCYL,4′-(4-dimethylaminophenylazo)benzoic acid (DABSYL),4-dimethylaminophenylazophenyl-4′-maleimide (DABMI),tetramethylrhodamine, carboxytetramethylrhodamine (TAMRA), BHQ™ dyes andthe like. Methods of coupling fluorophores and quenchers to nucleicacids are well known in the art.

In one embodiment of the present invention, the probes are molecularbeacon probes. As is known in the art, certain criteria need to be metfor a molecular beacon probe to be successful in monitoring or detectingan amplification reaction. The present invention, therefore, providesmolecular beacon probes that comprise polynucleotides of the presentinvention together with flanking self-complementary regions. Thepolynucleotides of the present invention may make up the loop region ofthe molecular beacon, or they may make up the loop region and part ofthe stem region. Thus, the self-complementary stem sequences can beunrelated to the target sequence or may contain one or more nucleotidesthat are complementary to the target sequence.

In one embodiment of the present invention, polynucleotides having anucleic acid sequence as set forth in any one of SEQ ID NOs: 7 or 12, orhomologues of these polynucleotides, together with appropriateself-complementary flanking sequences are provided as molecular beaconprobes. In a related embodiment, the molecular beacon probes have anucleic acid sequence as set forth in any one of SEQ ID NOs: 7 or 12.

One skilled in the art will understand that the selection of primers tobe used with the molecular beacon probe also requires certain criteriato be met. For example, it is important that there are no areas ofcomplementarity that may cause the molecular beacon to bind to a primer,which would result in a high background signal.

The polynucleotides according to the present invention, therefore, arefurther provided in combinations, comprising two primers and at leastone probe, that can be used to specifically amplify and detect targetnucleic acid sequences in a test sample. In a related embodiment,primer/probe sets are provided for the amplification and detection oftarget nucleic acid sequences by molecular beacon PCR.

As is known in the art, molecular beacon probes can be used to monitorthe progress of an amplification reaction in real time. During thecourse of an amplification reaction, such as a PCR, the molecular beaconinteracts with its target sequence at the annealing temperature for theprobe, and a fluorescent signal is generated. As the number of targetstrands produced in the amplification reaction increases, the number ofmolecular beacons bound to their target increases concomitantly, as doesthe strength of the fluorescent signal.

In accordance with the present invention, therefore, the combinations oftwo primers and at least one probe, as described above, can be used ineither end-point amplification and detection assays, in which thestrength of the detectable signal is measured at the conclusion of theamplification reaction, or in real-time amplification and detectionassays, in which the strength of the detectable signal is monitoredthroughout the course of the amplification reaction.

Patients potentially infected with N. gonorrhoeae are often also at riskfor infection with Chlamydia trachomatis. CT primers may be used inconjunction with primers for the amplification of NG to amplify targetsequences from either or both organisms in a single amplificationreaction. The co-amplification reaction, followed by species-specifichybridization reactions, allow for concurrent assessment of a testsample for infection with either CT, or NG, or both organisms. Accordingto another embodiment of the invention, both CT and NG can be amplifiedand/or detected simultaneously in a single reaction mixture using acombination of two primer/probe sets (i.e. one selected from the CTspecific primer/probe sets and the other selected from the NG specificprimer/probe sets). For example, a test sample could be contacted withprimer/probe sets 7 and 12, or with primer/probe sets 2 and 10, alongwith amplification reagents to amplify and detect the presence of CT andNG in a test sample. It is understood by one skilled in the art thatwhen two or more distinct target sequences are to be detected in thesame reaction mixture, each probe must be detectably distinct from eachof the other probes. For example if two or more molecular beacon probesare present in the same reaction mixture, the fluorophore moieties ofeach beacon probe should fluoresce at different wavelengths.

The polynucleotides according to the present invention can also be usedin assays to quantitate the amount of CT and/or NG nucleic acid presentin a sample. Thus, the polynucleotides according to the presentinvention can be used in a method to specifically amplify, detect andquantitate target nucleic acid sequences in a test sample, whichgenerally comprises the steps of:

-   (a) forming a reaction mixture comprising nucleic acid amplification    reagents, at least one polynucleotide probe sequence that    incorporates a label which produces a detectable signal upon    hybridisation of the probe to its target sequence, at least one    polynucleotide primer and a test sample that contains one or more    target nucleic acid sequences;-   (b) subjecting the mixture to amplification conditions to generate    at least one copy of the target nucleic acid sequence, or a nucleic    acid sequence complementary to the target sequence;-   (c) hybridising the probe to the target nucleic acid sequence or the    nucleic acid sequence complementary to the target sequence, so as to    form a probe:target hybrid;-   (d) detecting the probe:target hybrid by detecting the signal    produced by the hybridised labeled probe; and-   (e) comparing the amount of the signal produced to a standard as an    indication of the amount of target nucleic acid sequence present in    the test sample.

One skilled in the art will understand that, as outlined above, step (b)of the above method can be repeated several times prior to step (c) bythermal cycling the reaction mixture by standard techniques known in theart.

Various types of standards for quantitative assays are known in the art.For example, the standard can consist of a standard curve compiled byamplification and detection of known quantities of CT or NG nucleicacids under the assay conditions. Alternatively, an internal standardcan be included in the reaction. Such internal standards generallycomprise a control target nucleic acid sequence and a controlpolynucleotide probe. The internal standard can optionally furtherinclude an additional pair of primers. The primary sequence of thesecontrol primers may be unrelated to the polynucleotides of the presentinvention and specific for the control target nucleic acid sequence.Alternatively, no additional primer need be used if the control targetsequence is designed such that it binds at one end with a primer from afirst primer/probe set directed to a first target sequence (for example,CT), and binds at the other end with a primer for a second primer/probeset directed to a second target sequence (for example, NG), such thatcopies will be generated under amplifying conditions.

In the context of the present invention, a control target nucleic acidsequence is a nucleic acid sequence that:

-   (a) can be amplified either by a CT or NG primer or primer pair    being used in a particular reaction or by distinct control primers;-   (b) specifically hybridises to the control probe under suitable    conditions; and-   (c) does not hybridise with a CT- or NG-specific probe under the    same conditions.

In the context of the present invention, in addition to fulfilling thestandard requirements for probe molecules, the control polynucleotideprobe for use in quantitation reactions:

-   (a) specifically hybridises to the control sequence under suitable    conditions;-   (b) does not hybridise with a CT sequence, to the CT-specific probe,    or to the CT-specific primers under the same conditions, when a CT    target sequence is being detected;-   (c) does not hybridise with a NG sequence, to the NG-specific probe,    or to the NG-specific primers under the same conditions, when a NG    target sequence is being detected;-   (d) incorporates a detectable label that is distinct from the label    incorporated into other probes (for example CT and/or NG probes) in    use in the same reaction mixture. The signals generated by these    various labels when they bind their respective target sequences can    thus be distinguished and quantified separately.

One skilled in the art will recognise that the actual nucleic acidsequence of the control target nucleic acid and the control probe is notimportant provided that they both meet the criteria outlined above.

In the context of the present invention, the amount of target nucleicacid in a test sample can be quantified using “end point” methods or“real time” methods. One skilled in the art will appreciate that whenused as CT- or NG-specific probes in quantitative assays, thepolynucleotides of the present invention can be conventionalhybridisation probes, linear BHQ™ probes, TaqMan® probes, molecularbeacon probes, or combinations or modified versions thereof. In oneembodiment of the present invention, the polynucleotides are used asmolecular beacon probes.

The present invention also contemplates the provision of any one or moreof the polynucleotides of the invention, for example any one of theprimer sets or primer/probe sets of the invention together with acontrol target nucleic acid sequence, which can be amplified by thespecified primer pair, and a control polynucleotide probe for thequantitative reactions. The present invention further provides for theinclusion of control primers, which specifically amplify the controltarget nucleic acid sequence, in the quantitative reactions.

The amplification and/or detection methods in which the polynucleotidesaccording to the present invention can be employed are suitable foradaptation as high-throughput assays. High-throughput assays provide theadvantage of processing many samples simultaneously and significantlydecrease the time required to screen a large number of samples. Thepresent invention, therefore, contemplates the use of thepolynucleotides of the present invention in high-throughput screening orassays to detect and/or quantitate CT and/or NG nucleic acids in aplurality of test samples.

For high-throughput assays, reaction components are usually housed in amulti-container carrier or platform, such as a multi-well microtiterplate, which allows a plurality of assay reactions containing differenttest samples to be monitored in the same assay. The present inventionalso contemplates highly automated high-throughput assays to increasethe efficiency of the screening or assay process. Many high-throughputscreening or assay systems are now available commercially, as areautomation capabilities for many procedures such as sample and reagentpipetting, liquid dispensing, timed incubations, formatting samples intomicroarrays, microplate thermocycling and microplate readings in anappropriate detector, resulting in much faster throughput times.

The polynucleotides in accordance with the present invention can beprovided as part of a kit that allows for the detection and/orquantitation of CT and/or NG nucleic acids. Such kits comprise one ormore of the polynucleotides of the invention for use as a primer and/orprobe. In one embodiment of the present invention, the polynucleotidesare provided in the kits in combinations for use as primers tospecifically amplify CT and/or NG nucleic acids in a test sample. In arelated embodiment, the polynucleotides are provided in combinationsthat comprise the nucleic acid sequences as set forth in SEQ ID NOs: 1and 4; and/or SEQ ID NOs: 8 and 9.

In another embodiment, the polynucleotides are provided in the kits incombinations comprising at least two primers and at least one probe. Ina related embodiment, the polynucleotides are provided in combinationsthat comprise the nucleic acid sequences as set forth in SEQ ID NOs: 1,4, and 5; SEQ ID NOs: 2, 4, and 5; SEQ ID NOs: 3, 4, and 5, SEQ ID NOs:1, 4, and 6; SEQ ID NOs: 2, 4, and 6; SEQ ID NOs: 8, 9 and 10; SEQ IDNOs: 8, 9 and 11; or SEQ ID NOs: 8, 9 and 12. Kits comprisingcombinations of more than one primer/probe set are also envisaged, e.g.,without limitation, SEQ ID NOs: 1, 4, 5, 8, 9 and 10; and SEQ ID NOs: 2,4, 5, 8, 9 and 10.

Kits for the detection of CT and/or NG nucleic acids may additionallycontain a control target nucleic acid and a control polynucleotideprobe. Thus, in one embodiment of the present invention, the kitscomprise one of the above combinations of polynucleotides comprising atleast two primers and at least one probe, together with a control targetnucleic acid sequence, which can be amplified by the specified primerpair, and a control polynucleotide probe. The present invention furtherprovides kits that include control primers, which specifically amplifythe control target nucleic acid sequence.

The kits can optionally include amplification reagents, reactioncomponents and/or reaction vessels. Typically, at least one sequencebears a label, but detection is possible without this. Thus, one or moreof the polynucleotides provided in the kit may have a detectable labelincorporated, or the kit may include reagents for labeling thepolynucleotides. One or more of the components of the kit may belyophilised and the kit may further comprise reagents suitable for thereconstitution of the lyophilised components. The kit can additionallycontain instructions for use.

The polynucleotides, methods, and kits of the present invention areuseful in clinical or research settings for the detection and/orquantitation of CT and/or NG nucleic acids. Thus, in these settings thepolynucleotides can be used in assays to diagnose CT and/or NG infectionin a subject, or to monitor the quantity of a CT and/or NG targetnucleic acid sequence in a subject infected with CT and/or NG.Monitoring the quantity of bacteria in a subject is particularlyimportant in identifying or monitoring response to anti-bacterialtherapy.

Primers and probes useful for amplifying and/or detecting Chlamydiatrachomatis (CT) or Neisseria gonorrhoeae (NG) are presented below inTable 1.

TABLE 1  Polynucleotide Sequence (5′-3′) SEQ ID NO CT Forward primerGGGATTCCTG TAACAACAAG TCAGG 1 Alternative CT GGGATTCDTG TAACAACAAG TCAGG2 Forward primer (wherein D is not C) Preferred embodimentGGGATTCGTG TACAACAAG TCAGG, 3 of SEQ ID NO: 2 CT Reverse primerGCTTGCACGA AGTACTCTAG GAG 4 CT Probe ATAGCACTAT AGAACTCTGC AA 5Alternative CT Probe CATAGCACTA TAGAACTCTG CAAGCC 6 CT Beacon probectggcATAGC ACTATAGAAC TCTGCAAgcc ag 7 NG Forward primerCGACGTACCG GTTTTTGTTC 8 NG Reverse primer CGGCTCCTTA TTCGGTTTGA CC 9NG Probe ACACCGCCCG GAACCCGA 10 Alternative NG ProbeGAAACACCGC CCGGAACCCG AT 11 NG Beacon probe ctcggACACC GCCCGGAACC CGAg12

In Table 1, polynucleotide probe sequences complementary to thebacterial sequences are indicated in capital letters; lower casenucleotides are self-complementary so as to form a stem of a beaconprobe under suitable conditions; and in some instances, nucleotidescomplementary to the bacterial sequences also form part of theself-complementary beacon probe stem.

The nucleic acids having polynucleotide sequences of Table 1 can becombined in suitable combinations to form a primer/probe set useful inthe context of the present invention. Suitable primer probe setsinclude, but are not limited to:

-   -   Primer and Probe Set 1: SEQ ID NOs: 1, 4, and 5;    -   Primer and Probe Set 2: SEQ ID NOs: 2, 4, and 5;    -   Primer and Probe Set 3: SEQ ID NOs: 3, 4, and 5;    -   Primer and Probe Set 4: SEQ ID NOs: 1, 4, and 6;    -   Primer and Probe Set 5: SEQ ID NOs: 2, 4, and 6;    -   Primer and Probe Set 6: SEQ ID NOs: 3, 4, and 6;    -   Primer and Probe Set 7: SEQ ID NOs: 1, 4, and 7;    -   Primer and Probe Set 8: SEQ ID NOs: 2, 4, and 7;    -   Primer and Probe Set 9: SEQ ID NOs: 3, 4, and 7;    -   Primer and Probe Set 10: SEQ ID NOs: 8, 9, and 10;    -   Primer and Probe Set 11: SEQ ID NOs: 8, 9, and 11; and    -   Primer and Probe Set 12: SEQ ID NOs: 8, 9, and 12.

The following examples are for illustrative purposes only and should notbe construed to limit the scope of this invention in any way.

EXAMPLES

The following examples demonstrate detection of various subtypes of CTand NG using the primer/probe sets described above. These DNA primersand probes comprising the primer/probe sets are identified as SEQ IDNOs: 1-7 are specific for CT, and SEQ ID NOs: 8-12 are specific for NG.

In the following examples, SEQ ID NOs: 1 and 4 are used as consensusamplification primers specific for the CT target sequence. SEQ ID NO: 6or 7 is used as consensus internal hybridization probes for the CTamplification product. SEQ ID NOs: 8 and 9 are used as consensusamplification primers specific for the NG target sequence and SEQ ID NO:10 or 12 is used as consensus internal hybridization probes for the NGamplification product.

Example 1 Preparation of Primers and Probes

A. CT and NG Consensus Primers

Consensus primers were designed to detect all known CT or NG subtypes byoligonucleotide hybridization PCR. In the following examples, theseprimers were SEQ ID NO: 1 and SEQ ID NO: 4 for CT, and SEQ ID NO: 8 andSEQ ID NO: 9 for NG. SEQ ID NO: 3 was found to have some advantagesrelative to SEQ ID NO: 1 for some embodiments of the present invention,which relate to ease of purification of these polynucleotides. However,those embodiments are not illustrated below, and the inventors preferuse of SEQ ID NO: 1 to use of SEQ ID NO: 3.

B. CT and NG Consensus Probes

Consensus probes were designed to hybridize with the amplified CT or NGtarget sequence by oligonucleotide hybridization. These probe sequenceswere modified for use as probes in a molecular beacon assay by theaddition of terminal nucleotides to generate self-complementary 5′ and3′ ends, and of fluorescent (fluo) and quenching moieties at oppositeends of the polynucleotide (see Table 2). Thus the CT probe sequence ofSEQ ID NO: 6 was the basis of the molecular beacon probe of SEQ ID NO:7, and the NG probe sequence of SEQ ID NO: 10 was the basis of themolecular beacon probe of SEQ ID NO: 12. Where more than one beaconprobe was used simultaneously in the same molecular beacon assay,fluorescing moieties with different excitation-emission profiles wereused to distinguish between the different probe signals.

Probe sequences were synthesized using standard oligonucleotidesynthesis methodology.

TABLE 2  Sequence (5′-3′) CT beacon probeFluor-ctggcATAGC ACTATAGAAC TCTGCAAgcc ag-quencher (SEQ ID NO: 7)NG beacon probe Fluor-ctcggACACC GCCCGGAACC CGAg-quencher(SEQ ID NO: 12)

Example 2 Preparation of Sample DNA

CT DNA was isolated from CT serotype Ba, Apache-2 strain (ATCC VR-347.Rockville, Md.) using methods known in the art, and used to assess thesensitivity, specificity and cross-reactivity of the CT consensusprimer/probe sets. NG DNA was similarly prepared and used to assess thesensitivity, specificity and cross-reactivity of the CT consensusprimer/probe sets.

In negative control samples (“Neg”), an equal volume of diluent wassubstituted for sample DNA. Various concentrations of sample DNA of aknown concentration were included as positive control (“0.12×”, “1×”,“10×”).

An internal control (IC) target polynucleotide was designed as positivecontrol for amplification, for use with CT and NG primers. The sequencetargeted for amplification was complementary to a CT primer at one endand a NG primer at the other end, such that in the presence of bothprimers the control sequence would be amplified under amplifyingconditions. The polynucleotide was designed to yield an ampliconcomplementary to a control probe, for example a molecular beacon probe,but not complementary to the probes in use for the detection of eitherCT or NG. This control target polynucleotide was prepared using methodswell known in the art.

Example 3 Sensitivity of CT Detection

CT DNA was prepared as described in Example 2. Primer/probe set 7 wasevaluated (i.e., SEQ ID NOs: 1 and 4 were used as primers and SEQ ID NO:7 as the probe). All sequences were prepared as described in Example 1.

Dilutions of the isolated CT DNA were PCR amplified and detected usingprimer/probe set 7. PCR was performed in 1× GeneAmp® PCR Gold Buffer,0.25 mM EDTA and 0.125 mM EGTA. Amplitaq Gold™ DNA polymerase was usedat a concentration of 10 units/reaction, with dNTPs (dATP, dGTP, dTTPand dCTP) present at a final concentration of 0.20 mM each. A finalconcentration of 8 mM MgCl₂ was used in a total reaction volume of 0.1ml. Sample volumes were 50 containing none or 10′-10⁶ Chlamydialelementary bodies (EB)/reaction. Primers were used at a concentration of200-400 nM each, and beacon probes at a concentration of 100 nM each. Aninternal control (IC) target sequence and control probe were present ata final concentration of 500 copies/reaction and 100 nM respectively.

Reaction mixtures were amplified in a Perkin-Elmer Thermal Cycler.Reaction mixtures were first incubated at 95° C. for 9.5 minutes. PCRamplification was then accomplished in 45 cycles with each cycle being95° C. for 30 seconds then 59° C. for 60 seconds. After the reactionmixtures were thermal cycled, the mixtures were maintained at 95° C. for3 minutes and then lowered to 25° C. for 10 minutes for probehybridization. Reaction products were detected on a robotic microplatefluorescence reader using the following emission maxima and band widths(in nm):

TABLE 3 1^(st) Fluor 2d Fluor 3d Fluor Excitation 485/20 534/8 585/8Emission 517/8 562/12 612/12

Data from this experiment is presented in TABLE 4 and shows detection ofCT serotype Ba at concentrations as low as 10 EB/reaction usingprimer/probe set 3.

TABLE 4 [CT] (EB/reaction) Measured Fluorescence 0 2559 10 10441 10012451 1000 12760 10,000 14819 1,000,000 17243

Thus, this Example shows good target response over a range of 10 to 10⁶EB/reaction.

Example 4 Cross-Reactivity Between CT and NG

To test for cross-reactions between CT and NG primer/probe sets, targetDNA and primer/probe sets for both CT and NG were combined in eachreaction mixture to simultaneously detect CT and NG DNA in test samples.CT and NG DNA were prepared as in Example 2, and the samples prepared asoutlined below. The CT primer/probe set #7 (SEQ ID NOs: 1 and 4 asprimers and SEQ ID NO: 7 as probe) and NG primer/probe set #12 (SEQ IDNOs: 8 and 9 as primers and SEQ ID NO: 12 as probe) were prepared asdescribed in Example 1 and used either together or separately to amplifyand detect the dilutions of CT and NG DNA.

The samples included 2 cross-reactivity panels (samples 1-58 and samples1B-23B) as well as negative (Neg) and positive controls (0.12×, 1× and10×). Each reaction mixture comprised a final concentration of 0.40 uMCT forward primer (SEQ ID NO: 1), 0.20 uM CT reverse primer (SEQ ID NO:4), 0.10 uM CT beacon probe (SEQ ID NO: 7), 0.20 uM NG forward primer(SEQ ID NO: 8), 0.20 uM NG reverse primer (SEQ ID NO: 9), 0.10 uM NGbeacon probe (SEQ ID NO: 12), 5 units of Amplitaq DNA Polymerase, 1 mMdNTPs, and 8 mM MgCl₂ in 1×PCR Buffer II. 100 ul reactions were cycled45 times through 95° C./30 sec and 59° C./1 min for DNA amplification,followed by 1 cycle at 95° C./3 mM and 25° C./10 min for finaldenaturation and probe annealing.

Importantly, detection of CT was not altered by the inclusion of the NGprimer/probe set with the CT primer/probe set in the CT reactionmixtures, with sensitivity remaining at approximately 10′ organisms perreaction. Similarly, detection of NG was not altered by the inclusion ofthe CT primer/probe set with the NG primer/probe set in the NG reactionmixtures, with sensitivity remaining at approximately 10′ organisms perreaction.

The NG assay reagents detected NG, but did not detect any of over 50other non-sexually transmitted (non-STD) strains of Neisseria, nor 3strains of Chlamydia (trachomatis, pneumoniae, and psittaci).

Example 5 CT Serovar Detection

To determine whether and with what sensitivity primers and probes of theinvention could detect a range of CT serovars, a dilution panel of 15 CTserotypes was tested in reactions that included primer/probe sets forboth CT and NG, and suitable negative (Neg) and positive (1× and 0.12×)control samples. The CT primer/probe set #7 and NG primer/probe set #12were prepared as described in Example 1, and the CT and NG control DNAwere prepared as in Example 2.

The CT serovar samples were present in the reaction mixtures at finalconcentrations in the range of 10-1000 molecules per reaction. Eachreaction mixture comprised a final concentration of 0.40 uM CT forwardprimer (SEQ ID NO: 1), 0.20 uM CT reverse primer (SEQ ID NO: 4), 0.10 uMCT beacon probe (SEQ ID NO: 7), 0.45 uM NG forward primer (SEQ ID NO:8), 0.25 uM NG reverse primer (SEQ ID NO: 9), 0.10 uM NG beacon probe(SEQ ID NO: 12), 3 units of Amplitaq DNA Polymerase, 1 mM dNTPs, and 8mM MgCl₂ in 1×PCR Buffer II. 100 ul reactions were cycled 45 timesthrough 95° C./30 sec and 59° C./1 min for DNA amplification, followedby 1 cycle at 95° C./3 mM and 25° C./10 min for final denaturation andprobe annealing.

Sixteen serovars of Chlamydia trachomatis were detected usingprimer/probe sets of the present invention over concentrations of10-1000 copies per reaction.

Example 6 NG Auxotype Detection

To determine whether and with what sensitivity primers and probes of theinvention could detect a range of NG subtypes, a dilution panel of 7 NGauxotypes was tested in reactions that included primer/probe sets forboth CT and NG, and suitable negative (Neg) and positive (1× and 0.12×)control samples. The CT primer/probe set #7 and NG primer/probe set #12were prepared as described in Example 1, and the CT and NG control DNAwere prepared as in Example 2.

The NG auxotype samples were present in the reaction mixtures at finalconcentrations in the range of 10-1000 molecules per reaction. Eachreaction mixture comprised a final concentration of 0.40 uM CT forwardprimer (SEQ Ill NO: 1), 0.20 uM CT reverse primer (SEQ ID NO: 4), 0.10uM CT beacon probe (SEQ ID NO: 7), 0.20 uM NG forward primer (SEQ ID NO:8), 0.20 uM NG reverse primer (SEQ ID NO: 9), 0.10 uM NG beacon probe(SEQ ID NO: 12), 3 units of Amplitaq DNA Polymerase, 1 mM dNTPs, and 8mM MgCl₂ in 1×PCR Buffer II. Reactions were cycled 45 times through 95°C./30 sec and 59° C./1 min for DNA amplification, followed by 1 cycle at95° C./3 min and 25° C./10 mM for final denaturation and probeannealing.

Six auxotypes of NG were tested, and all tested auxotypes were detectedwith high sensitivity (10 copies per reaction) using the primer/probesets of the invention. In addition, no cross reactivity was observedwith the CT primer set even when NG was present at a high concentration(10⁷ NG molecules per reaction).

All publications, references, patents and patent applications referredto above are specifically incorporated by reference to the same extentas if each reference were individually incorporated by reference in itsentirety

While the invention has been described in detail and with reference tospecific embodiments, it will be apparent to one skilled in the art thatvarious changes and modifications may be made to such embodimentswithout departing from the spirit and scope of the invention.

What is claimed is:
 1. A composition comprising three polynucleotidesconsisting of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 12, wherein thepolynucleotide of SEQ ID NO: 12 is labeled with a detectable label.
 2. Aprimer/probe set consisting of polynucleotides of SEQ ID NOs: 8, 9, and12, wherein the polynucleotide of SEQ ID NO: 12 is labeled with adetectable label.
 3. A method of amplifying Neisseria gonorrhoeae in atest sample, said method comprising: (a) forming a reaction mixturecomprising nucleic acid amplification reagents, a test samplepotentially containing a Neisseria gonorrhoeae target sequence acomposition according to claim 1; and (b) subjecting the mixture toamplification conditions to generate at least one copy of a nucleic acidsequence complementary to the target sequence.
 4. The method of claim 3wherein the amplification conditions include changing the temperature ofthe sample and the temperature change is repeated between 10 and 100times.
 5. A method of detecting Neisseria gonorrhoeae in a test sample,said method comprising: (a) forming a reaction mixture comprisingnucleic acid amplification reagents, a test sample potentiallycontaining a Neisseria gonorrhoeae target sequence, and (b) subjectingthe mixture to amplification conditions to generate at least one copy ofa nucleic acid sequence complementary to the target sequence; (c)hybridizing the probe to the nucleic acid sequence complementary to thetarget sequence, so as to form a hybrid comprising the probe and thenucleic acid sequence complementary to the target sequence; and (d)detecting the hybrid as an indication of the presence of Neisseriagonorrhoeae in the test sample.
 6. The method of claim 5 wherein saidreaction mixture further comprises a control target polynucleotide and acontrol polynucleotide probe.
 7. A kit comprising: (a) the primer/probeset of claim 5; and (b) amplification reagents.
 8. The kit of claim 7further comprising: (a) two or more polynucleotides each having adistinct nucleic acid sequence selected from the group consisting of SEQID NOs: 2, 4, 5, 6, 7, 10, and
 11. 9. A method of detecting Neisseriagonorrhoeae in a test sample, said method comprising: (a) forming areaction mixture comprising nucleic acid amplification reagents, a testsample potentially containing a Neisseria gonorrhoeae target sequence,and a primer/probe set according to claim 5; (b) subjecting the mixtureto amplification conditions to generate at least one copy of a nucleicacid sequence complementary to the target sequence; (c) hybridizing theprobe to the nucleic acid sequence complementary to the target sequence,so as to form a hybrid comprising the probe and the nucleic acidsequence complementary to the target sequence; and (d) detecting thehybrid as an indication of the presence of Neisseria gonorrhoeae in thetest sample.